In recent years, structural and functional analyses of proteins in living tissues have been rapidly promoted as post-genome research. As one method for such structural and functional analyses of proteins (proteome analysis), the methods that involve using a mass spectrometer for the expression analysis or primary structure analysis of a protein have been widely used in recent years. One of these commonly known methods uses a mass spectrometer capable of MSn analysis (n≧2) including the operations of selecting and dissociating a specific ion. According to this method, the amino acid sequence of a protein is determined as follows.
First, a protein of interest is digested with an appropriate enzyme into a mixture of peptide fragments, and this peptide mixture is subjected to mass analysis. The elements constituting those peptides include stable isotopes having different masses. Therefore, in the aforementioned mass analysis, even a group of peptides consisting of the same amino acid sequence produce a plurality of peaks having different m/z values due to their difference in isotope composition. These peaks include a peak corresponding to the “main” ion, which consists of only the isotope having the highest natural abundance ratio, and one or more peaks corresponding to the “isotope” ions, which contain an isotope in addition to the most abundant isotope. When these ions are monovalent, they form an “isotope peak group”, i.e. a group that consists of a plurality of peaks arranged at intervals of one Da to several Da.
Subsequently, among a set of mass spectrum data of the aforementioned peptide mixture, one isotope peak group originating from one peptide is selected as precursor ions, and a mass analysis of the ions (product ions) obtained by dissociating these precursor ions (i.e. an MS/MS analysis) is performed. By searching a database for the mass-spectrum pattern of the obtained product ions or the mass-spectrum pattern of the precursor ions, it is possible to determine the amino acid sequence of the peptide being examined and identify the protein concerned (for example, refer to Patent Document 1).
The previously described protein identification method basically assumes that the sample is prepared by extracting a protein from a cell or other living tissues and then purifying and separating the protein. However, in the field of biochemistry or medicine, there are extremely strong demands for obtaining information about the two-dimensional distribution of proteins inside the cell in a living organism without destroying the cell whenever possible. To meet such demands, intensive efforts have been made on the development of a mass microscope (which is also referred to as an imaging mass spectrometer), which is capable of functioning both as a microscope and as a mass spectrometer. Mass microscopes can obtain distribution information (or a mapping image) of a substance within a two-dimensional area on a sample which is set, for example, on a preparation. Several configurations have been proposed to obtain mass-spectrum data for each micro area within a two-dimensional area on a sample in the mass microscope.
For example, in mass spectrometers disclosed in Patent Document 2, Patent Document 3 and Non-Patent Document 1, the irradiation point of a laser beam or particle beam for ionizing a sample is sequentially moved on the sample, and the ions generated from the irradiation point are individually detected for each m/z value every time the irradiation point is changed. In a mass spectrometer disclosed in Non-Patent Document 2, ions are almost simultaneously generated in a two-dimensional form so that they reflect the two-dimensional distribution of a substance on the sample. Then, those ions are separated for each m/z value by a time-of-flight mass separator and detected by a two-dimensional detector.
In any of the aforementioned configurations, in order to obtain a mapping image of a substance present within a two-dimensional area on a sample, it is necessary to analyze and process mass-spectrum data obtained for each micro area within the two-dimensional area to identify a substance (typically, a protein) present within each micro area. In the case of the mass spectrometer capable of MS/MS analysis, a set of mass-spectrum data obtained as a result of a mass analysis without dissociating an ion is analyzed and processed to determine an ion to be selected as a precursor, after which an MS/MS analysis is performed for each micro area with an appropriate precursor selected for the micro area and a set of MS/MS spectrum data obtained by the MS/MS analysis is analyzed and processed to identify the substance present within the micro area.
As a display form for showing a result based on the mass-spectrum data or MS/MS spectrum data obtained for each of the micro areas in the previously described manner, the following two examples are commonly known (for example, refer to Non-Patent Document 3).
(A) A mass spectrum of a measurement point (to be exact, a micro area that has an extremely small area and can be regarded as a point) on the sample or an average mass spectrum obtained by averaging mass spectrums of a plurality of points is displayed on a screen. An operator visually checks the mass spectrum and specifies an m/z range to be observed. Then, a mapping image is created on the display screen, on which the spectrum intensity value of the specified m/z range at each measurement point within a two-dimensional area on the sample is shown by a specific color pattern.
(B) On an optical image of a sample surface or a mapping image showing a two-dimensional distribution of a specific m/z value (or m/z range), an operator sets an ROI (region of interest) frame of an arbitrary shape to specify a portion to be observed. Then, the average of the mass spectrums of a plurality of measurement points included in the range surrounded by the ROI frame is calculated, and the thereby created average mass spectrum is drawn on the display screen.
The technique (A) provides information about the m/z value of a substance that is spatially localized on a sample. Therefore, for example, it is possible to know the m/z value of a substance that is not present in the nose or chin but localized in the brain or a specific portion of the brain. The technique (B) facilitates the comparison of mass spectrums obtained at different spatial areas of a sample. Therefore, this technique is convenient, for example, when the mass spectrums of the brain, nose, chin or other portions are to be compared.
The techniques (A) and (B) reveal the distribution of a substance but do not identify the localized substance. Therefore, although an operator can recognize a certain substance as the substance to be observed (which is hereinafter called the substance of interest) but cannot specifically discern the kind of the substance of interest. To identify the substance of interest, it is necessary to do a more complex procedure, for example as follows: After a portion where the substance of interest is present is located by technique (A), the operator specifies that portion by setting an ROI frame on an optical image or mapping image by technique (B), and enters a command to initiate an MS/MS analysis with the m/z value of the substance of interest as the precursor. Then, MS/MS spectrums obtained at a plurality of measurement points by the measurement are averaged to obtain an average MS/MS spectrum. Using the information of a peak or peaks appearing on this average mass spectrum, a commonly known database search is performed to identify the substance of interest.
The previously described procedure is troublesome and complex for an operator, and hence inefficient and time consuming. Furthermore, since the range specified by the ROI frame on the optical image or mapping image cannot be reduced to an adequately small size, the MS/MS spectrums used for the calculation of the average MS/MS spectrum inevitably include many peaks originating from unwanted substances other than the substance of interest. These noises deteriorate the S/N ratio of the MS/MS spectrums, making it difficult to improve the identification accuracy.    Patent Document 1: Japanese Unexamined Patent Application Publication No. 2006-284509    Patent Document 2: Specification of U.S. Pat. No. 5,808,300    Patent Document 3: Japanese Unexamined Patent Application Publication No. 2007-66533    Non-Patent Document 1: Kiyoshi OGAWA et al., “Kenbi Shitsuryou Bunseki Souchi No Kaihatsu (Research and Development of Mass Microscope)”, Shimadzu Hyouron (Shimadzu Review), Shimadzu Corporation, published on Mar. 31, 2006, Vol. 62, Nos. 3 and 4, pp. 125-135    Non-Patent Document 2: Yasuhide NAITO, “Seitai Shiryou Wo Taishou Ni Shita Shitsuryou Kenbikyou (Mass Microprobe Aimed at Biological Samples)”, J. Mass Spectrom., Soc. Jpn., Vol. 53, No. 3, 2005    Non-Patent Document 3: “msimaging BioMap”, MS imaging, [search Jul. 3, 2008], Internet